Avian and mammalian receptors for 1,25-dihydroxyvitamin D₃: In vitro translation to characterize size and hormone-dependent regulation

In vitro translation of cellular poly(A)⁺ RNA coupled with immunoprecipitation was developed as a technique for characterizing 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptors and assessing receptor mRNA activity. Cell-free translation of poly(A)⁺ RNA isolated from chicken intestine revealed two immunoprecipitable forms of avian receptor at 60 kDa and 58 kDa. These two species were identical in electrophoretic mobility to those detected directly in intestinal cytosol by immunoblot analysis. Liver, a tissue devoid of 1,25-(OH)₂D₃ binding activity, contained no apparent translatable receptor mRNA. 1,25-(OH)₂D₃ receptors were also synthesized in vitro employing poly(A)⁺ RNA obtained from several cultured mammalian cell lines. Selective immunoprecipitation revealed a single form of receptor at 54 kDa in mouse fibroblasts (3T6) and pig kidney cells (LLC-PK₁) and a 52-kDa species in human breast carcinoma (T47D). Each of these in vitro translated mammalian 1,25-(OH)₂D₃ receptors migrated identically with its cellular counterpart that was synthesized in vivo employing metabolic labeling of cell protein with [³⁵S]methionine. In vitro translation of poly(A)⁺ RNA derived from mouse 3T6 cells treated with 1,25-(OH)₂D₃ for 24-48 hr disclosed a 5-fold increase in receptor mRNA activity over untreated control cells. These results are consistent with the conclusions that 1,25-(OH)₂D₃ receptors are protein species ranging from 52 to 60 kDa and that, though their functional and immunological domains have been evolutionary conserved, an inverse relationship apparently exists between phylogenetic status and receptor mass. The data also support the hypothesis that the presence of 1,25-(OH)₂D₃ leads to a significant increase in receptor mRNA activity in 3T6 cells, indicative of receptor autoregulation.